Bacillus anthracis is a spore-forming bacterium that is the causative agent of anthrax, a disease that often attacks lungs or connective tissue. Cutaneous anthrax is the most common form of the disease which causes skin redness, boils or ulceration after B. anthracis spores infect injured skin or membranes and germinate to vegetative cells that produce anthrax toxin. If bacteremia develops, it may lead to high fever and death. The respiratory form occurs following inhalation of spores which infect alveolar macrophages and germinate in lymph nodes causing edema, hemorrhaging, lymph node necrosis and pleural effusion. Respiratory anthrax, which is usually fatal within a week of infection, may lead to septicemia and meningitis. Oropharyngeal and gastrointestinal anthrax are the least common forms of the disease, which usually result from ingestion of contaminated meat that has been insufficiently cooked. Although uncommon and beginning with nonspecific symptoms (sore throat, fever, nausea, vomiting), these forms of anthrax have about a 50% mortality rate. Although anthrax usually attacks herbivorous animals, humans who come in contact with contaminated animal hair, wool, hides, meat, or waste can contract the disease. Human infection caused by deliberate release of B. anthracis spores, resulting in cutaneous and/or respiratory anthrax, is a form of biological warfare or bioterrorism. Infected humans can be treated for anthrax by using antibiotics, e.g., penicillin. Accurate detection of the presence of B. anthracis and diagnosis of infection is important because of the high fatality rate of the disease and the risk to others who may be exposed to an infected individual or contaminated items.
B. anthracis is related to other species in the Bacillus genus, all of which are endospore forming bacteria that are gram-positive rods. B. anthracis is closely related to commonly occurring non-anthrax species, such as B. cereus, B. cereus var mycoides, B. thuringiensis, B. megaterium and B. subtilis. Because of the close genetic relationships of B. cereus, B. cereus var mycoides, B. anthracis, and B. thuringiensis, these species are often grouped as members of the B. cereus complex, or even considered one species (Kaneko et al., 1978, Microbiol Immunol. 22: 639-41; Helgason et al., 2000, Appl. Environ. Microbiol. 66: 2627-30). For example, this relationship is exhibited in the gene sequences encoding 16S rRNA (1,554 nt), in which there are one to four nt differences between the genes of B. anthracis, B. cereus, and B. thuringiensis strains, although the B. anthracis sequence was highly conserved for 86 strains tested and the B. thuringiensis sequence was highly conserved for 11 strains tested (Sacchi et al., 2002, Emerging Infect. Dis. 8(10): 1117-23).
Virulent strains of B. anthracis carry virulence genes on two plasmids, pXO1 and pXO2 (GenBank accession nos. AF065404 and AF188935, respectively). Genes on plasmid pXO1 code for proteins that contribute to the toxicity of B. anthracis infection: protective antigen (PA), edema factor (EF), and lethal factor (LF). PA is a membrane-binding protein required for toxicity when combined with either EF or LF. The gene pag codes for PA, the gene cya codes for EF, and the gene lef codes for LF (Price et al., 1999, J. Bacteriol. 181(8):2358-2362). Genes on plasmid pXO2, designated capB, capC, and capA, code for elements of the antiphagocytic capsule of B. anthracis. Known strains of B. anthracis retain both, one or none of these plasmids. Generally, a strain that has lost one or both of these plasmids is considered avirulent. A virulent strain contains both plasmids, such as the Ames (pXO1+/pXO2+) strain. An avirulent strain that has lost the pXO2 plasmid is strain Δ Sterne (pXO1+/pXO2−), spores of which are used worldwide as a live vaccine for animals (Hambletone et al., 1984, Vaccine 2: 125-32). An avirulent strain that has lost the pXO1 plasmid is strain Δ Ames (pXO1−/pXO2+). Strains that have lost both plasmids are known, e.g., strain VNR1-Δ1.
Clinical identification of B. anthracis relies on procedures such as microscopic examination of Gram-stained smears made from a specimen or cultured bacteria, detection of nonhemolytic or weakly hemolytic growth on blood agar, observation of granular or ground-glass texture of colonies grown for greater than 36 hrs on agar media, or mucoid colonies which are associated with virulent encapsulated forms (Logan et al., In: Manual of Clinical Microbiology, 7th Ed., Murray et al., eds., 1999, American Society for Microbiology (Washington, D.C.), pp. 357-69). Under microscopic examination, B. anthracis cells are large rods, usually in chains and encapsulated, which generally are nonmotile compared to motile strains of B. cereus and B. thuringiensis. B. anthracis rods may or may not contain oval central or subterminal spores, which form when the bacteria are exposed to low CO2 levels such as found in the atmosphere. Clinical testing for B. anthracis includes preparing and examining laboratory cultures made from the sample, e.g., inoculating broth and blood cultures and streaking on laboratory media such as 5% sheep blood agar, MacConkey agar, and phenyl ethyl alcohol agar. Cultures are incubated for at least 3 days and observed daily beginning as early as 8 hours after inoculation to determine the growth characteristics of the bacteria or colonies. While hemolysis, gram stain morphology, or motility results can be used to rule out the presence of B. anthracis, a combination of two test results is recommended to rule out B. anthracis as the organism present in the tested sample. Because other members of the B. cereus complex group may mimic B. anthracis in appearance and characteristics, interpreting test results is challenging and definitive identification of B. anthracis from culture is difficult. Additional assays for identification of B. anthracis have relied on detecting encapsulated organisms by using antibodies, and detecting α-glucosidase activity. These tests usually require at least 3 days to rule out the presence of B. anthracis or for positive identification of the Bacillus species because of the relatively long growth period of colonies in the laboratory. Moreover, clinical laboratories that perform these tests require equipment to permit procedures be done at a biosafety level of 2 (BSL-2) or greater. If a clinical laboratory is unable to rule out the presence of B. anthracis or definitively identify the Bacillus species, then the sample is referred to another laboratory for further testing.
In addition to these clinical tests, other methods may be used to detect and identify Bacillus species, including B. anthracis (Harrell et al., 1995, J. Clin. Microbiol. 33: 1847-1850; Keim et al., 1999, J. Appl. Microbiol. 87(2): 215-7; Jackson et al., 1997, Appl. Environ. Microbiol. 63(4): 1400-5; Sirard et al., 2000, Int. J. Med. Microbiol., 290(4-5): 313-6); Mock et al., 2001, Ann. Rev. Microbiol. 55: 647-71). For example, B. anthracis has been detected by using tests based on the polymerase chain reaction (PCR) to amplify bacterial nucleic acid sequences that identify B. anthracis (Makino et al., 1993, J. Clin. Microbiol. 31(3): 547-51; Ramisse et al., 1996, FEMS Microbiol. Lett. 145(1): 9-16; Makino et al., 2001, Lett. App. Microbiol. 33(3): 237-40; Keim et al., 1997, J. Bacteriol. 179(3): 818-24; Patra et al., 1998, J. Clin. Microbiol. 36(11): 3412-14; Daffonchio et al., 1999, Appl. Environ. Microbiol. 65(3): 1298-303; Lee et al., 1999, J. Appl. Microbiol. 87(2): 218-23; Fasanella et al., 2001, Vaccine 19(30):4214-18; Enserink, 2001, Science 294(5545): 1266-7). Another method of detecting B. anthracis relies on detecting B. anthracis-specific polymorphic signature sequences isolated from chromosomal DNA (Rastogi et al., U.S. Pat. No. 6,448,016 B1). One assay relies on amplifying and detecting species-specific fragments of the gyrB genes to distinguish between B. cereus, B. thuringiensis, and B. anthracis (Yamada et al., U.S. Pat. No. 6,087,104).
Because of the clinical significance of anthrax infections, and the additional psychological and economic impacts of bioterrorism threats or events that may involve B. anthracis, there remains a need for effective methods to detect and identify B. anthracis that may be present in environmental and clinical samples (Lane and Fauci, 2001, JAMA 286:2596-7; Enserink, 2001, Science 294(5545): 1266-7). There is a particular need to identify and distinguish virulent forms of B. anthracis from non-virulent B. anthracis or other similar Bacillus species. Initial testing of samples in the United States may be done at a Level A clinical laboratory (of the Laboratory Response Network for Bioterrorism (“LRN”), categorized by the Centers for Disease Control and Prevention (“CDC”), Atlanta, Ga., USA) that is not be equipped or trained to identify Bacillus species, and then samples suspected of containing B. anthrax are referred to other laboratories (e.g., LRN Level B or C) for further testing. To avoid delays in identification of the Bacillus species or unnecessary referrals to higher level laboratories for identification, there remains a need for an assay that quickly allows a Level A laboratory to at least rule out the presence of B. anthracis from a tested sample and more preferably to identify the presence of B. anthracis in a sample.